Sustained-release microparticle preparation of human growth hormone and process for producing thereof

ABSTRACT

Provided is a sustained-release microparticle preparation of a human growth hormone which combines biodegradability with sustained-release performance while avoiding the use of an organic solvent as much as possible, allows the sustained-release of a human growth hormone over 3 days or more and reduction in the initial burst release, can contain a human growth hormone at 10% or more, can quantitatively adsorb and encapsulate a human growth hormone at up to 20%, and has excellent dispersion and needle penetration properties. A process for producing the sustained-release microparticle preparation of a human growth hormone is also provided. The sustained-release microparticle preparation comprises a porous apatite derivative, a human growth hormone, and a water-soluble divalent metal compound.

TECHNICAL FIELD

The present invention relates to a sustained-release microparticlepreparation of a human growth hormone comprising, as a base, fineparticles of a porous apatite derivative that is bioerodible in theliving body and to a process for producing THEREOF.

BACKGROUND ART

Investigation has heretofore been made on injections that provide thesustained release of a water-soluble drug for a long time, most of whichcomprise poly-lactic-co-glycolic acid (PLGA) as a base (see e.g.,Japanese Patent Laid-Open Nos. 11-286403, 2000-239104, and 2002-326960).Alternatively, sustained-release microcapsules that contain a humangrowth hormone (hGH) and comprise PLGA as a base have been reported (seee.g., Nature Medicine, 2: 795-799, 1996). Sustained-releasemicrocapsules that contain leuprorelin, a LHRH agonist, and comprisePLGA as a base have also been reported (see e.g., ChemicalPharmaceutical Bulletin, 36: 1095-1103, 1988). PLGA is a biodegradablebase that hydrolyzes and eliminates in the living body, and thisproperty is preferable for a base of an injection.

Although an organic solvent that dissolves PLGA is generally used forproducing a sustained-release preparation with PLGA, hGH is denatured inthe organic solvent and a portion thereof is inactivated. Such reductionin activity not only impairs efficacy but poses the risk of adverselyaffecting a living body. Furthermore, hGH is highly soluble in water,and the use of a PLGA preparation inevitably results in the excessiverelease of hGH in the early stage after administration. In addition,although the use of hydrogel or the like has been reported, the hydrogelis difficult to administer by means of a typical injection. That is, athick needle that permits the gel to pass therethrough must be used andis unfavorable for patients. Sustained-release particles usinghydroxyapatite and a human growth hormone that is a bioactive drug havealready been reported (see e.g., H. Gautier et al., Journal ofBiomedical Material Research, 40,606-613,1998; and J. Guicheux et al.,Journal of Biomedical Material Research, 34, 165-170, 1997). However,any of the sustained-release particles are two-component systems whereapatite has a large particle size of 40 to 80 μm or 200 μm and istherefore difficult to inject. Moreover, their in vivo sustained-releaseeffect is unknown. Besides, the amount of hGH adsorbed into the apatiteparticle (the amount of hGH encapsulated) was as low as 1% or less.

For a sustained-release preparation of hGH, a material having so-calledbiodegradability or a property of eliminating in the living body, whicheliminates from a living body about the end of the release of a drugafter administration, must be selected as a sustained-release base. Inits production, a sustained-release preparation is prepared with the useof an organic solvent avoided as much as possible for preventing thedenaturation of hGH. At the same time, the initial burst release (therelease of an excessive drug in the early stage after administration) ofhGH must be small. Moreover, the amount of a drug encapsulated in amicroparticle preparation is brought to 10% or more by weight. Thepreparation capable of being easily administered with a thin injectionneedle must be prepared, wherein the duration of the sustained-releaseof a drug extends at least 3 days or more.

The present inventors have found that the use of fine particles of aporous apatite derivative for solving these problems achieves apreparation combining biodegradability with sustained-releaseperformance, without the use of an organic solvent. The presentinventors have further found that the use of hGH in combination with awater-soluble divalent metal compound achieves the sustained-release ofhGH over at least 3 days or more and reduces initial burst release. Inaddition, the present inventors have found that a hGH content of 10% ormore can be attained and hGH up to 20% can quantitatively be adsorbedand contained, and also found that the obtained fine granularpreparation has excellent dispersion and needle penetration propertiesand easily passes through a 27 G injection needle.

Thus, an object of the present invention is to provide asustained-release fine granular preparation of a human growth hormonewhich combines biodegradability with sustained-release performance whileavoiding the use of an organic solvent as much as possible, allows thesustained release of a human growth hormone over 3 days or more andreduction in the initial burst release, can contain a human growthhormone at 10% or more, can quantitatively adsorb and include a humangrowth hormone at up to 20%, and has excellent dispersion and needlepenetration properties. The present invention is also intended toprovide a process for producing the sustained-release fine granularpreparation of a human growth hormone.

DISCLOSURE OF THE INVENTION

A sustained-release microparticle preparation of a human growth hormonecomprises a porous apatite derivative, and a human growth hormone and awater-soluble polyvalent metal compound contained in the porous apatitederivative. The water-soluble polyvalent metal compound includes zincchloride, calcium chloride, calcium hydroxide, iron chloride, ironhydroxide, cobalt chloride, aluminum chloride, and zinc acetate. Amongothers, a water-soluble divalent metal compound is most preferable. Thewater-soluble divalent metal compound is preferably a zinc compound anda calcium compound, most preferably zinc chloride. In addition, zincacetate, calcium chloride, and so on are preferable.

It is preferred that the porous apatite derivative should be a porousapatite derivative in which a portion of calcium as a constituent ofhydroxyapatite is substituted by zinc.

It is also preferred that the substitutional atomic ratio of zinc tocalcium in the porous apatite derivative should be 0.1 to 2.0.

Moreover, it is preferred that the porous apatite derivative should havea human growth hormone content of 10 to 40%.

A process for producing the sustained-release microparticle preparationof a human growth hormone of the present invention comprises: stirringand dispersing fine particles of a porous apatite derivative in watercontaining a human growth hormone; after the water is infiltrated intothe porous apatite derivative, adding an aqueous solution containing awater-soluble divalent metal compound to infiltrate the water-solubledivalent metal compound into the porous apatite derivative; and thenadding an additive such as a stabilizer, followed by freeze-drying orvacuum-drying.

The sustained-release microparticle preparation of hGH thus obtained isa sustained-release microparticle preparation of hGH comprising a porousapatite derivative (a porous apatite derivative in which a portion ofcalcium as a constituent of hydroxyapatite is substituted by any ofother metals, preferably by zinc) slowly soluble in water, hGH, and anaqueous divalent metal compound. More preferred is the sustained-releasemicroparticle preparation of hGH in which the ratio of zinc to calciumin the porous apatite derivative soluble in water is 0.1 to 2.0, whilethe water-soluble divalent metal compound is a zinc compound.

In a basic process for producing the sustained-release microparticlepreparation of hGH, fine particles of a porous apatite derivative aredispersed in water containing hGH and, after the water is sufficientlyinfiltrated into the porous apatite derivative, an aqueous solutioncontaining a water-soluble divalent metal compound is added tosufficiently infiltrate the water-soluble divalent metal compound intothe porous apatite derivative. Then, an appropriate additive such as astabilizer is added, followed by freeze-drying or vacuum-drying tothereby obtain a powder of a sustained-release microparticle preparationcomprising, as a base, the porous apatite derivative containing the hGH.When actually administered, this powder is dispersed in an appropriatedispersion medium and administered, for example, subcutaneously orintramuscularly.

When the desorption of the encapsulated hGH is measured by dispersingthe microparticles thus obtained in a large amount of purified water atroom temperature, the hGH of an amount below a certain amount withrespect to porous apatite derivatives was not desorbed, depending on thetypes of the porous apatite derivatives. That is, the hGH is probablyinfiltrated and adsorbed into the fine pores of the porous apatitederivatives.

The fine particles of a porous apatite derivative used in the presentinvention can be obtained by a known method. The method includes amethod described in, for example, T. Yamaguchi, H. Yanagida, A.Makishima, H. Aoki, Ceramic Science Series 7 Bioceramics, GIHODO SHUPPANCo., Ltd., pp. 7-9, 1984. The most preferable porous apatite derivativeis any of those in which a portion of Ca is substituted by zinc (Zn) inthe composition of hydroxyapatite. The ratio of zinc which substitutescalcium in hydroxyapatite (the ratio of Zn atoms substituted to Ca 10atoms of hydroxyapatite) is preferably 0.1 to 5.0, more preferably 0.5to 2.0. In this case, the velocity of the hydroxyapatite eliminating inthe living body differs depending on the ratio of (Ca+Zn)/P. If theratio is smaller than 1.67, the hydroxyapatite is more likely to besoluble in water, that is, the velocity of the hydroxyapatiteeliminating in the living body is accelerated. It is preferred that theratio of (Ca+Zn)/P should fall within the range of 1.67 to 1.51. A lowertreatment temperature at which hydroxyapatite is produced renders thehydroxyapatite more soluble in water, that is, accelerates the velocityof the hydroxyapatite eliminating in the living body. The treatmenttemperature used is room temperature to 800° C., preferably 150° C. to600° C. More preferable is a treatment temperature of 150° C. to 400° C.If hydroxyapatite is treated at 800° C. or higher, the hydroxyapatitedoes not eliminate in the living body. If hydroxyapatite is treated at100° C. or lower, particles thereof tend to agglomerate together and aretherefore difficult to administer by a typical injection. The particlesize of each fine particle can be controlled by a treatment temperatureand can be used in the range of 0.1 μm to 100 μm. Of these sizes,preferred is 0.1 μm to 20 μm. The fine particles having a size of 0.2 μmto 10 μm can be preferably used.

A porous apatite derivative having a larger ratio of Zn whichsubstitutes calcium in the porous apatite derivative to the porousapatite derivative had a much larger ratio of hGH adsorbed into theporous apatite derivative. This is attributed to the specific surfaceand porosity of the porous apatite derivative that are significantlyincreased by allowing zinc to be substituted. The larger ratio of hGHadsorbed is preferred because the gross amount of the sustained-releasepreparation administered is rendered smaller. The preferred ratio of hGHadsorbed differs depending on the duration of the sustained-release ofthe hGH and however, is generally 2 to 30% by weight with respect to theporous apatite derivative. Of these rates, 5 to 25% by weight ispreferably used. More preferred is any of those adsorbing 10% or more byweight of hGH therein.

The water-soluble divalent metal compound that is added after the hGH isadsorbed into the porous apatite derivative is preferably a Zn or Cacompound. Of them, the Zn compound is most preferable. The amount of itsusage is preferably in the range of 10 to 100% by weight on a zincchloride basis with respect to the porous apatite derivative and morepreferably in the range of 20 to 70% by weight for sufficientlymaintaining the sustained-release property. A chloride or a salt of anorganic acid is preferably selected as the water-soluble divalent metalcompound used. Examples thereof include zinc chloride, zinc acetate, andcalcium chloride.

The particle size of the sustained-release microparticle preparationfinally obtained may be a size that allows the preparation to passthrough an injection needle used in typical administration. In reality,the smaller size an injection needle has, the less a patient is scared.It is preferred that the sustained-release microparticle preparationshould pass through an injection needle with a thickness of 25 G orsmaller (the greater the number is, the thinner an injection needlegets) defined by the international standard that specifies the thicknessof an injection needle. It is more preferable that the sustained-releasemicroparticle preparation should pass through an injection needle with athickness of 27 G or smaller. For this reason, a sustained-releasemicroparticle preparation having a smaller particle size is morepreferable. However, a sustained-release microparticle preparationhaving a particle size rendered small to the extreme reduces the amountof a drug retained therein and the increases initial burst release of adrug. Thus, the particle size is preferably 0.5 μm to 20 μm, morepreferably 0.5 μm to 10 μm. The duration of the sustained release of thehGH-containing sustained-release microparticle preparation thus obtainedcan be controlled by a temperature at which the hydroxyapatite(HAP)derivative is treated and the amount of usage of the divalent metalcompound, allowing the sustained -release of hGH over 3 days or more.The sustained-release of hGH over 1 week or more is made possible and ispreferred in practical ways.

EXAMPLES

The present invention will be described hereinafter in detail withreference to Examples and however, is not intended to be limited tothese Examples by any means.

Example 1

To 50 mg of a porous HAP or a porous apatite derivative (HAP-Zn-0.5: 0.5parts zinc atom with respect to 9.5 parts calcium atom) treated at 180°C., a human growth hormone (hGH) solution (25 mg/ml) desalted with aPD-10 column (Amersham Pharmacia) was added (200 μL to the HAP and 700μL to the HAP-Zn-0.5) and stirred for 1 minute with a touch mixer. Next,water was added to each of the solutions to adjust the final volume to2.5 mL and further stirred for 1 minute. After being left undisturbedfor 3 minutes, the mixtures were centrifuged at 3,000 rpm for 3 minutes,and the resulting precipitations were supplemented with 2.5 mL of water,respectively, and stirred for 1 minute. The mixtures were centrifugedagain at 3,000 rpm for 3 minutes, and the resulting precipitations weresupplemented with 0.333 mL (25 μmol of zinc chloride), 1 mL (75 μmol ofzinc chloride), and 2 mL (150 μmol of zinc chloride) of an aqueoussolution of 10.2 mg/mL zinc chloride (Wako Pure Chemical Industries,Ltd., Osaka, Japan), respectively, and stirred with a touch mixer,followed by freeze-drying to prepare samples in a powder form. A sampleused as a control was prepared by adding water instead of a zincchloride solution. The volume mean diameters of the obtained samplesranged from 6 to 8 μm with the maximum being 20 μm. hGH contents in theobtained HAP-hGH and HAP-Zn-0.5-hGH samples were quantified with themicro BCA protein assay kit (Pierce). In addition, the in vitro releaseproperties of the obtained samples were compared. The release propertieswere evaluated by the following procedures: 5 mg of each of the obtainedsamples was accurately weighed, then supplemented with 0.250 mL of PBS(phosphate buffered saline), and stirred at 37° C.; the supernatantswere periodically collected by centrifugation at 3,000 rpm for 3minutes, and the amounts of hGH released into the supernatants werequantified by gel filtration HPLC analysis (TOSO G2000SW-x1) tocalculate the percentage of the amount of hGH released to the grossamount of hGH contained in each of the samples. This result is shown inTable 1. The samples supplemented with zinc chloride had remarkablereduction in the amount of hGH released into PBS in response to theamount of zinc chloride added, as compared to the sample prepared as acontrol in which only hGH was allowed to act on HAP.

TABLE 1 Effect of zinc chloride on in vitro release property ofmicroparticle preparations of porous apatites adsorbing hGH thereinPercentage of cumulative amount of hGH released into PBS to gross amountof hGH (%) 1 hr 2 hr 4 hr 24 hr HAP-hGH-no zinc chloride 35 37.7 38.843.9 HAP-hGH-zinc chloride- 0 0 0.093 0.27 25 μmol HAP-hGH-zincchloride- 0 0 0 0 75 μmol HAP-hGH-zinc chloride- 0 0 0 0 150 μmolHAP-Zn-hGH-zinc chloride- 0 0 0 0 188 μmol

Example 2

To 10 mg of each of three porous apatite derivatives in which a portionof calcium in HAP was substituted by zinc (HAP-Zn-0.1:0.1 parts zincatom with respect to 9.9 parts calcium atom; HAP-Zn-0.5: 0.5 parts zincatom with respect to 9.5 parts calcium atom; and HAP-Zn-1.0:1.0 partszinc atom with respect to 9.0 parts calcium atom) and HAP (all of whichare treated at 180° C.), 50 μL, 100 μL, 200 μL, and 400 μL of a hGHsolution (10 mg/ml) desalted with a PD-10 column (Amersham Pharmacia)were added, respectively, and stirred for 1 minute with a touch mixer.Water was further added to each of the solutions to adjust the finalvolume to 500 μL and stirred for 1 minute with a touch mixer. Afterbeing left undisturbed for 3 minutes, the mixtures were centrifuged at3,000 rpm for 3 minutes, and the amounts of hGH contained in theresulting precipitations were quantified with the micro BCA proteinassay kit (Pierce) to compare the amount of hGH in each of the samples.This procedure allows the detection of only hGH adsorbed into the porousapatite derivatives for each of the samples. The result is shown inTable 2. The volume mean diameter of the obtained samples ranged from 6to 8 μm with the maximum being 20 μm. The porous apatite derivativesundergoing zinc substitution had significant increase in the amount ofhGH adsorbed, as compared to the HAP undergoing no zinc substitution.Moreover, the amount of hGH adsorbed greatly increased as the proportionof zinc introduced into the porous apatite derivative increased. When 10mg of the HAP-Zn-1.0 was used and the amount of hGH charged is not morethan 2 mg, the whole hGH charged was adsorbed therein. At this time, ahGH content is 20%.

TABLE 2 Comparison of amounts of hGH adsorbed into porous apatitederivatives Amount of hGH Amount of hGH adsorbed (mg/HAP 10 mg) charged(mg) HAP HAP-Zn-0.1 HAP-Zn-0.5 HAP-Zn-1.0 0.5 0.51 0.53 0.52 0.51 1 0.961.05 1.06 1.02 2 1.25 1.49 1.87 1.92 4 1.52 1.94 2.74 2.91

Example 3

To 50 mg of HAP or HAP-Zn-0.5, a hGH solution (10 mg/ml) was added (0.5mL to HAP and 1.75 mL to HAP-Zn-0.5) and stirred for 1 minute with atouch mixer. Next, water was added to each of the solutions to adjustthe final volume to 2.5 mL and further stirred for 1 minute with a touchmixer. After being left undisturbed for 3 minutes, the mixtures werecentrifuged at 3,000 rpm for 3 minutes, and the resulting precipitationswere supplemented with 2.5 mL of water, respectively, and stirred againfor 1 minute. The mixtures were centrifuged again at 3,000 rpm for 3minutes, and the resulting precipitations were supplemented with 1 mL(75 μmol) (for HAP-hGH) and 2.25 ml (188 μmol) (for HAP-Zn-hGH) of aaqueous solution of 10.2 mg/mL zinc chloride (Wako) and stirred with atouch mixer, followed by freeze-drying to prepare samples (representedby HAP-hGH-zinc chloride and HAP-Zn-hGH-zinc chloride, respectively). Asample used as a control is prepared by adding water instead of a zincchloride solution (represented by HAP-hGH or HAP-Zn-hGH). All of thevolume mean diameters of the obtained samples ranged from 6 to 8 μm whenmeasured with a coulter counter. hGH contents in the obtained HAP-hGHand HAP-Zn-hGH samples were quantified with the micro BCA protein assaykit (Pierce) The HAP-hGH, the HAP-hGH-zinc chloride, and theHAP-Zn-hGH-zinc chloride were suspended in 0.5% CMC-Na, 5% mannitol, and0.1% Tween 80. And 10 IU/kg (1 IU: 0.35 mg) of each resulting suspensionwas subcutaneously administered to the back of a male SD rat. Inaddition, hGH dissolved in the same solvent was administered as acontrol.

Table 3 shows the dispersion and needle penetration properties of eachof the preparations at the time of administration. The HAP-hGH had poordispersion property and did not pass through a 27 G injection needle. Incontrast, the preparations supplemented with the HAP-Zn-hGH and zincchloride were improved in dispersion and needle penetration properties.Concerning a needle penetration properties for a 27 G injection needle,particularly the HAP-Zn-hGH-zinc chloride passed through the 27 Ginjection needle more easily than the other two preparations that passedtherethrough.

After 1, 2, 4, and 8 hours post-administration and subsequently on every1 day, blood was collected from the tail vein the rats to measure theblood level of hGH using the E test “TOSOH” II (HGH) (a full automaticEIA device AIA-6000, TOSOH Corp). This result is shown in Table 4. Whena hGH solution was administered, hGH quickly eliminated from blood andno hGH was detected in blood after 8 hours post-administration. In thecase of the HAP-hGH, high burst was observed in the early stage. On theother hand, in the case of the HAP-hGH-zinc chloride, initial burst wasconsiderably reduced, and sustained-release effect was observed over 1week. The HAP-Zn-hGH-zinc chloride was confirmed to have increase in ahGH content and increase in the sustainability of the blood level ofhGH. Moreover, when the preparation remaining in the hypodermis on 20days post-administration was visually observed, it was observed that themost parts thereof disappeared.

TABLE 3 Comparison of dispersion needle penetration properties ofmicroparticles preparations of porous apatites containing hGH Needlepenetration hGH content Dispersion properties (% by weight) property 23G25G 27G HAP-hGH 7.6 X ◯ Δ X HAP-Zn-hGH 22.3 ◯ ◯ ◯ ⊚ HAP-hGH-zinc 5.9 ◯ ◯◯ ◯ chloride HAP-Zn-hGH-zinc 16.2 ⊚ ◯ ◯ ⊚ chloride

TABLE 4 Time courses in blood level of hGH after subcutaneousadministration of fine granular preparations of porous apatites to ratshGH content in preparation Blood level of hGH (ng/mL) (% by weight) 1 hr2 hr 4 hr 8 hr 1 day 2 day 3 day 4 day 5 day 6 day 7 day hGH 249 139 0.50 HAP-hGH 7.6 301 326 224 46.3 2.8 0.9 0.37 0.14 0.11 0.05 0.02HAP-Zn-hGH 22.3 343 294 170 27.3 1.41 0.86 0.35 0.15 0.08 0.02 0.02HAP-hGH-zinc 6.6 5 16.9 51.3 99.7 11.1 2.03 1.49 0.65 0.4 0.16 0.2chloride HAP-Zn-hGH-zinc 16.2 4 11.7 26.4 49.1 11.8 2.81 1.32 0.78 0.430.32 0.18 chloride (Average value of n = 3 for each time)

1. A sustained-release microparticle preparation of human growth hormonecharacterized in that human growth hormone and a water-soluble divalentmetal compound are adsorbed to a zinc-containing porous hydroxyapatitewhich is formed by partially substituting calcium atoms of poroushydroxyapatite with zinc.
 2. The sustained-release microparticlepreparation of a human growth hormone according to claim 1,characterized in that the water-soluble divalent metal compound is zincchloride.
 3. The sustained-release microparticle preparation of a humangrowth hormone according to claim 1, characterized in that the number ofatoms of zinc contained in the zinc-containing porous hydroxyapatite is0.1 to 2.0 relative to 10 atoms of calcium of the porous hydroxyapatite.4. The sustained-release microparticle preparation of a human growthhormone according to claim 1, characterized in that the content of humangrowth hormone in the zinc-containing porous hydroxyapatite is 10 to 40%by weight.